The six-protein shelterin complex cooperates with telomerase to maintain the integrity of chromosomal ends. As part of shelterin, the protein ACD/TPP1 protects telomeric ends from the DNA damage response machinery. Additionally, ACD/TPP1 is uniquely involved in recruiting telomerase to telomeres through a dedicated protein surface known as the TEL patch. Both end-protection and end-elongation are important for the maintenance and function of somatic stem cells in aging tissues. We showed that ACD/TPP1 has essential context- specific functions in hematopoietic stem cells and in other tissues. Recent work identified candidate ACD/TPP1 mutations in patients with Dyskeratosis congenita, a human disorder characterized by impaired somatic stem cell function in the bone marrow and other organs, with features of premature aging. This is the first identification of ACD/TPP1 mutations in human disease. Our preliminary data show that these mutations can disrupt telomerase recruitment and telomere homeostasis in cell lines. However, how telomere maintenance defects in cells due to ACD/TPP1 mutations translate to dysfunction of specific stem cell compartments in vivo and how they contribute to tissue aging and disease is not understood. We hypothesize that ACD/TPP1 mutations can play a causative role in Dyskeratosis congenita and related somatic stem cell disorders. Furthermore, we postulate that point mutations affecting the interface of ACD/TPP1 with its protein partners can reveal unique functions of the shelterin complex when modeled in mammalian tissues. To explore this hypothesis in detail, we will study candidate mutations and other variants affecting binding of ACD/TPP1 with telomerase or with its shelterin partner TIN2, using modern genome-editing technology to assess their in vivo effects in the entire organism. Our Specific Aims are to: 1) Evaluate the impact of candidate human ACD/TPP1 TEL patch mutations on hematopoietic stem cells and bone marrow homeostasis; 2) Determine the structural requirements for the interaction of ACD/TPP1 with TIN2 and the functional consequences of defective TIN2- ACD/TPP1 binding; 3) Investigate how tissue homeostasis and longevity are affected by candidate ACD/TPP1 pathogenic variants that disrupt interaction with key protein partners. Our multidisciplinary approach will identify distinct functional surfaces of ACD/TPP1 and determine their impact on stem cells, tissue homeostasis and aging. We anticipate that we will uncover how mutational dysregulation of ACD/TPP1 and the shelterin/telomerase complex can ultimately result in human disease.